Protocol for studying topological DNA interactions by purified fission yeast condensin

Summary To understand the transition from interphase chromatin into well-shaped chromosomes during cell divisions, we need to understand the biochemical activities of the contributing proteins. Here, we present a protocol to investigate how the ring-shaped condensin complex sequentially and topologically entraps two DNA substrates. We describe the steps to prepare purified Schizosaccharomyces pombe condensin, as well as bulk biochemical assays to monitor the first and second DNA capture reactions. This protocol may facilitate further investigations of these essential genome organizers. For complete details on the use and execution of this protocol, please refer to Tang et al.1


SUMMARY
To understand the transition from interphase chromatin into well-shaped chromosomes during cell divisions, we need to understand the biochemical activities of the contributing proteins.Here, we present a protocol to investigate how the ring-shaped condensin complex sequentially and topologically entraps two DNA substrates.We describe the steps to prepare purified Schizosaccharomyces pombe condensin, as well as bulk biochemical assays to monitor the first and second DNA capture reactions.This protocol may facilitate further investigations of these essential genome organizers.For complete details on the use and execution of this protocol, please refer to Tang et al. 1

BEFORE YOU BEGIN
The protocol below describes the steps to investigate how the chromosomal condensin complex sequentially topologically entraps two double stranded DNA (dsDNA) substrates to establish dsDNA-dsDNA interactions.This protocol can be adapted to study related Structural Maintenance of Chromosome (SMC) complexes such as cohesin, the SMC5-SMC6 complex, or other proteins that might entrap more than one DNA substrate.
Before you begin, note that this protocol requires the use of pBlueScript supercoiled plasmid dsDNA as a DNA substrate.While the plasmid can be purified by various methods, we recommend using CsCl gradient centrifugation 2 as the DNA purification method for best results.Other purification methods, such as MiniPrep, will generate dsDNA plasmids with a mixture of supercoiled, nicked, and linear topologies.These plasmid preparations will become troublesome especially when you wish to study the impact of DNA topologies in the loading assays described later.
This protocol uses the budding yeast Saccharomyces cerevisiae as host cells to ectopically overexpress the fission yeast Schizosaccharomyces pombe condensin complex for purification.We observe that, of the five subunits that make up condensin, 3 the Cnd1 subunit is often present at somewhat substoichiometric levels when compared to the other four subunits in the purified condensin sample.As Cnd1 plays a key role in condensin's topological loading reaction onto DNA, we recommend selecting for a balanced subunit expression to generate condensin complexes with high specific activity and low non-specific DNA binding.While optimized wild type condensin expression strains have been described, 1 we describe the method to select such strains in the following to guide researchers to generate any variant condensin complexes in their own studies.

Construction of a budding yeast strain overexpressing Pk-epitope-tagged fission yeast condensin
Timing: 3-4 weeks 1.Clone the cDNA sequences of fission yeast condensin subunits into three integrative budding yeast plasmids under the control of the bidirectional GAL1-10 promoter.
Note: Each plasmid contains one or two of the fission yeast condensin subunits (Figure 1A).
2. Transform to integrate the three plasmids using the standard Lithium acetate protocol, 4 one at a time, into the genome of the W303 budding yeast strain lacking the abundant vacuolar protease (pep4D).
Note: pep4D strain can reduce unwanted protein degradation during cell lysis.

Note:
We transform the plasmids in the order specified below to facilitate tracking of protein expression during strain construction.

Note:
The Gal4 transcription factor is also expressed under galactose-inducible control, which aids expression of the multiple galactose-controlled condensin subunits.
Note: We recommend that the culture OD 600 should not exceed 1.5.c.Dilute the culture to OD 600 = 0.3 using fresh YPR and continue shaking at 25 C for 2 h.d.After the 2-h growth, note down an OD 600 reading and take 2 mL sample from the culture into a 2 mL screw cap cell breaker tube.Process sample according to the protocol below.
i. Centrifuge at 6,000 3 g for 1 min at room temperature (25 C).
iii.Resuspend cell pellet in 0.5 mL ice-cold 20% TCA (trichloroacetic acid) solution.iv.Incubate on ice for at least 10 min.e. Induce expression by addition of 2 mL filtered 20% galactose solution into the remaining culture.f.Continue shaking at 25 C for 4 h.g.After the 4-h induction, note down another OD 600 reading and take 2 mL sample from the culture.Process this sample as described in step 3d to fix cells in 20% TCA solution and incubate on ice for at least 10 min.h.Centrifuge the cell samples in 20% TCA, from before and after galactose induction, at 8,000 3 g for 1 min.Discard supernatant.i. Resuspend pellet in 0.5 mL 1 M Tris-HCl pH 8 solution.j.Centrifuge again at 8,000 3 g for 1 min.Discard supernatant.k.Resuspend the cell pellet in 30 mL SDS-PAGE sample buffer.l.Boil at 95 C for 5 min.m.Add 50 mL acid-washed glass beads.n.Rupture cells by vigorous shaking in a bead beater, e.g., MP Biomedicals FastPrep-24, using the default program for Saccharomyces cerevisiae cell lysis, i.e., 2 m/s speed for 40 s.
Note: Confirm efficient cell breakage by microscopy, adjust breakage cycles if required.
o. Puncture sample tube and collect lysate into a clean 1.5 mL Eppendorf tube by brief centrifugation as described. 5p.To adjust for the increased cell density following induction, dilute the induced sample 2-fold using SDS-PAGE loading buffer.q.Load 8 mL of the samples onto 10% Tris-Glycine SDS-PAGE for separation.r.Transfer the protein from SDS-PAGE gel onto a nitrocellulose membrane.s.Detect Cut14-Pk 3 expression using an a-Pk tag antibody (Figure 1B).
Note: Cnd2-3C-ProtA 2 will also be detected by virtue of its protein A tag.
Screening for stoichiometric condensin subunit overexpression using small-scale purification Timing: 1 day 4.After obtaining colonies from the third transformation, streak at least 10 colonies onto a new selection plate to increase chances of finding a suitable expression strain.5. Induce condensin expression from each colony in a small-scale culture.
a. Grow up each colony in 50 mL YPR medium at 25 C until OD 600 reaches between 0.6 and 1.  1C).Choose a colony that yields a stoichiometric condensin subunit composition, as indicated by five equal intensity bands following Coomassie Blue staining.11.Transfer the chosen expression strain as a 15% glycerol stock to À70 C for long term storage.Autoclave and store at room temperature (25 C) for a few months.

Timing: 2.5 days
This part of the protocol details the growth conditions to overexpress condensin and cell harvest.Note: We recommend growing up budding yeast freshly from the glycerol stock for largescale culture and subsequent protein purification.
2. On the next day, inoculate 50 mL YPD medium with the condensin-expressing strain and shake at 25 C for 4-8 h.
Note: Do not allow this pre-culture to grow beyond OD 600 = 1.5.
3. Inoculate 2 L YPR medium with the required pre-culture volume to reach an OD 600 of 0.6 on the next morning.
Note: We recommend inoculating the 2 L YPR medium on the night before and culturing the cells at 25 C overnight (between 12 -14 h) until the next morning.This will allow sufficient time for the yeast to adapt to the raffinose as the carbon source.

Note:
The doubling time of the condensin-expressing budding yeast is 2 h in our laboratory but may differ depending on local variation of media and growth conditions.Use the measured doubling time to calculate the amount of pre-culture to inoculate the 2 L cultures.We recommend inoculating four 2 L YPR cultures for better yield.
4. On the next day, when the OD 600 of the cultures reaches around 0.6, induce condensin overexpression by addition of 0.2 L filtered 20% galactose per 2 L culture. 5. Continue shaking the culture at 25 C for 4 h.6. Collect cells by centrifugation in a JLA8.1 rotor at 3500 rpm (or 3500 3 g) at 4 C for 10 min.

Note:
The centrifuge bottles can be reused to collect cell cultures.
7. Resuspend all of the cell pellet in 0.6 L ice-cold ddH 2 O. 8. Centrifuge again in a JLA8.1 rotor at 3500 3 g at 4 C for 10 min.9. Resuspend the cells in 50 mL IgG lysis buffer.10.Using a 10 mL pipette tip, drip the cell suspension drop by drop into liquid nitrogen.11.Collect the flash frozen cell suspensions in a beaker and store at À70 C.
Pause point: The flash-frozen yeast cells can be stored at À70 C for a few months.

Timing: 2 days
This part of the protocol details the condensin purification from the harvested yeast cells.Protocol 12. Break the cells by grinding under liquid nitrogen, e.g., using a Cole-Parmer cryogenic Freezer/ Mill using its standard protocol for yeast lysis (6 cycles of 1 min cooling and 2 min of cell grinding at 15 cps).

Note:
The tubes for the Freezer/Mill should not be more than two thirds filled.
13. Thaw the yeast powder at 4 C and mix well with 100 mL IgG lysis buffer.
Note: Ensure cells are completely thawed and no clumps remains.
14. Clear the lysate by centrifugation at 100,000 3 g (e.g., 35,000 rpm in a Beckman 45Ti rotor) at 4 C for 45 min.15.Carefully transfer the cleared lysate to a clean 200 mL Duran bottle.
Note: Avoid the white flurry layer at the top and the brown viscous layer at the bottom.16.Add 2 mL Rabbit IgG-Agarose beads (4 mL beads suspension) to the cleared lysate derived from 8 L culture.17.Seal the bottle with its cap and roll at 4 C for 1 h.18. Pour the incubated beads into a clean gravity flow column, e.g., Econo-Pac column from Bio-Rad.19.Wash the beads once with 20 mL IgG wash buffer.
Optional: After the first wash, we recommend washing the beads once with 10 mL IgG wash buffer supplemented with 10 mM MgCl 2 and 1 mM ATP.This wash helps remove chaperone proteins that might associate with the overexpressed condensin.20.Wash the beads six times with 20 mL IgG wash buffer.21.Resuspend beads in 20 mL IgG wash buffer and transfer to a clean 50 mL tube.22. Add to the suspension 40 mg 3C protease to cleave off the Protein A tag for protein elution and 40 mg RNase A to facilitate protein elution.Seal the tube and roll at 4 C overnight (16 h).23.Next day, pour the beads suspension onto a clean gravity column.Collect the flow through which contains the eluted condensin (Figure 2A).Troubleshooting 1. 24.Using a liquid chromatography system, e.g., AKTA system, load the supernatant onto a 1 mL Hi-Trap Heparin column, pre-equilibrated with Heparin A buffer.25.Wash the Heparin column with 20 mL Heparin A buffer.26.Elute condensin with a 20 mL linear gradient from Heparin A to Heparin B buffer.Collect 0.5 mL fractions.
Optional: In addition to taking samples from each fraction for SDS-PAGE analysis, we recommend testing each fraction for nuclease contamination.We incubate 10 mL from each fraction with 50 ng supercoiled plasmid, such as pBlueScript, in the presence of 10 mM MgCl 2 at 30 C for 1 h.Then each sample is supplemented with 0.5% SDS and 1 mg/mL Proteinase K followed by incubation at 50 C for 30 min.Finally, we separate DNA on a 0.8% agarose gel and stain with GelRed.Loss of the plasmid, or appearance of nicked or linear species, is indicative of contaminating nuclease activity.
27. Pool the peak fractions that are free of nuclease (Figure 2B).Troubleshooting 2. 28.Concentrate the condensin sample by ultrafiltration e.g., in a Vivaspin 6 concentrator, to 0.5 mL Troubleshooting 1. 29.Load the concentrated condensin sample onto a Superose 6 gel filtration column pre-equilibrated with Gel filtration buffer.Collect 0.25 mL fractions.30.Store the purified condensin.a. Pool the peak fractions (Figure 2C).b.Concentrate condensin, again by ultrafiltration, to around 1 mg/mL.c.Prepare aliquots and flash freeze those in liquid nitrogen.d.Store at À70 C until use.
Pause point: The flash-frozen purified condensin can be stored at À70 C for a few months.

Preparation of the digoxygenin-labeled linear dsDNA substrate
Timing: 6 h This step of the protocol details the preparation of digoxygenin-labeled dsDNA substrate (dig-DNA).
31.Preparation of the DNA substrate is based on triple digoxygenin-labeled oligonucleotide DNA primers (MT392 and MT393), ordered from a primer synthesis company, e.g., Integrated DNA Technologies.
CRITICAL: Each of the two primers should be triply digoxygenin-labeled for reliable binding to the beads.Singly digoxygenin-labeled primers were found to detach during incubations.32.PCR amplify the template plasmid (pEGFP-C1, ClonTech) with oligonucleotide primers MT462 and MT463, e.g., using CloneAmp PCR reagents according to manufacturer's instructions.33.After separating the amplified DNA on 0.8% Tris-Acetate-EDTA agarose gel, gel purify the amplified DNA, e.g., using Nucleospin gel and PCR cleanup according to manufacturer's instructions.34.PCR amplify the purified DNA product from the first round of amplification using digoxygeninlabeled oligos MT392 and MT393, e.g., using CloneAmp PCR reagents according to manufacturer's instructions.. 35.After separating the amplified DNA on 0.8% Tris-Acetate-EDTA agarose gel, gel purify the amplified DNA, e.g., using Nucleospin Gel and PCR cleanup according to manufacturer's instructions.36.Measure the yield and concentration using Nanodrop spectrophotometry.
Pause point: The purified dig-DNA (digoxygenin-labelled dsDNA) can be stored at À20 C for a year.

Condensin loading and second dsDNA capture
Timing: 4 h Note: This section of the protocol must be immediately followed by the protocols detailed in the next sections, which takes an additional 4-6 h.This step of the protocol details the condensin loading and second dsDNA capture reactions.We first couple the dig-DNA via a-digoxygenin antibodies to magnetic beads that can be easily washed and collected using the DynaMag magnet.We then perform the condensin loading reaction.After washing off excess condensin, we incubate the beads with pBlueScript plasmid to initiate the second dsDNA capture reaction.Finally, we use buffer with high ionic strength to remove condensin or pBlueScript plasmids that are not stably bound to the digoxygenin-labeled dsDNA on the beads (Figure 3A).
Note: Unless stated otherwise, all buffers in this step should be kept on ice throughout the experiment.37.For x number of loading reactions, pipette 10x mL protein G coupled Dynabeads suspension into a clean low-binding microcentrifuge tube.38.Wash the protein A coupled Dynabeads in 1 mL PBSA-BSA buffer twice.
Note: All subsequent steps regarding washing the protein A coupled Dynabeads refers to the following procedure.Resuspend beads in the indicated amount of buffer by carefully inverting the tubes multiple times until no clumps remain.Then immediately use a magnet to collect the Dynabeads at the side wall of the tube.Aspirate or pipette off the buffer.39.Resuspend the beads in 100x mL PBSA-BSA buffer and constantly rotate the tube at 4 C for 20 min.40.Mix 2x mL DNA binding buffer with 0.3x mL anti-digoxygenin antibody and 100x ng digoxygeninlabeled linear dsDNA.Incubate the reaction at room temperature (25 C) for 30 min.41.After incubation, add the antibody/DNA mix to the Dynabeads suspension and continue rotating the tube at 4 C for at least 1.5 h.42.Wash the beads once with 1 mL PBSA-BSA buffer, twice with 1 mL Loading wash buffer, and once with 1 mL Loading pre-equilibration buffer.43.Resuspend beads in 100x mL Loading pre-equilibration buffer and aliquot suspension into x lowbinding microcentrifuge tubes.
44. Collect beads using a magnet.Remove supernatant as much as possible.45.Resuspend beads in 15 mL Loading reaction buffer supplemented with 200 nM purified condensin and 1 mM ATP. 46.Incubate and shake the tubes with 850 rpm at 30 C for 30 min in a thermomixer.Completely resuspend the beads by tapping the tubes every 5 min to avoid beads aggregating at the bottom of the tubes.Troubleshooting 3.
Optional: After the incubation, if you are interested in the unbound fraction, collect beads using a magnet and take sample from the supernatant for DNA and/or protein analyses as detailed the section below.
47. Wash the beads three times with 1 mL Loading pre-equilibration buffer.48.Resuspend beads in 0.1 mL Loading pre-equilibration buffer.49.Collect beads using a magnet and discard the supernatant.50.Resuspend beads in 15 mL Loading reaction buffer supplemented with 1 mM ATP and 100 ng pBlueScript supercoiled dsDNA plasmid.51.Incubate and shake the tubes with 850 rpm at 30 C for 30 min in a thermomixer.Completely resuspend the beads by tapping the tubes every 5 min to avoid beads aggregating at the bottom of the tubes.
Optional: After the incubation, if you are interested in the unbound fraction, collect beads using a magnet and take sample from the supernatant for DNA and/or protein analyses as detailed the section below.
52. Wash the beads three times with 1 mL Loading wash buffer and once with 1 mL Loading preequilibration buffer.
Sample analyses after the second dsDNA capture reaction Timing: 4 h After the condensin second dsDNA capture reaction, immediately follow the protocol in this section to analyze the bead-bound protein and DNA components.
53. Resuspend beads in 1 mL Loading pre-equilibration buffer.a.Take 0.7 mL sample from the suspension for DNA analysis.b.Take 0.25 mL sample from the suspension for protein analysis.c.For both samples, collect beads using a magnet and remove the supernatant.54.To analyze protein content.a.To each sample, add 10 mL SDS sample buffer.b.Boil samples at 95 C for 5 min.c.Collect beads using a magnet and apply the supernatant for Tris-Glycine protein gel electrophoresis followed by Coomassie Blue staining.Troubleshooting 4. 55.To analyze DNA content.a.To each sample, add 10 mL Loading DNA elution buffer.b.Incubate samples at 50 C for at least 20 min with vigorous shaking.c.Collect beads using a magnet and load the supernatant for TAE-agarose gel electrophoresis followed by GelRed staining.Troubleshooting 5.

Restriction digestion of the reaction products Timing: 2 h
To investigate topology of the product after condensin second dsDNA capture reaction, we use restriction enzymes to specifically cut either the digoxygenin-labeled linear dsDNA or the supercoiled pBlueScript dsDNA plasmid, then analyze the DNA and protein contents on the beads and in the supernatant (Figure 3B).
56. Resuspend beads in 0.1 mL Loading pre-equilibration buffer.57.Collect beads using a magnet and resuspend beads in 10 mL 1x CutSmart buffer with or without 1 mL StuI or 1 mL ScaI-HF.58.Incubate the tubes at 18 C and shake at 1100 rpm in a thermomixer for 1 h.59.Cool the reaction on ice and add 10 mL 1x CutSmart buffer supplemented with 1 M NaCl.Resuspend well by tapping the tubes.60.Take 14 mL for DNA analyses and 5 mL for protein analyses.Collect the beads using a magnet and keep the supernatant for downstream analyses.
61. Analyze the DNA and protein samples according to steps detailed in the section above.

EXPECTED OUTCOMES
Before we begin, we construct a fission yeast condensin over-expression strain by integrating three plasmids (Figure 1A) sequentially into the budding yeast host.Since the protein A tag, which is fused to Cnd2 from the second plasmid, binds to any antibody, the integration of the first two plasmids can be confirmed simultaneously on Western blot of the whole cell extract after galactose induction of protein expression using an a-Pk antibody (Figure 1B).Integration and galactose-induced protein expression from the third plasmid must be confirmed using a small-scale IgG pull down.This step is crucial because of the various expression or subunit incorporation levels of Cnd1 among different clones (Figure 1C).
The final expression strain is then cultured in a large volume before galactose induction.Fission yeast condensin is then purified using a three-step protocol, consisting of IgG pull down, heparin affinity chromatography, and a Superose 6 size exclusion chromatography step.Small samples of protein can be collected from each step and analyzed by SDS-PAGE (Figure 2).From a typical 8 L cell culture harvested with a final OD 600 between 1.5 and 2, we expect to obtain between 200 mg to 300 mg purified fission yeast condensin complex at a concentration of over 1 mg/mL.
Using the purified condensin, we perform a DNA loading and second DNA capture reaction.We split the beads in our reactions to analyze both the DNA and protein content remaining on the beads after high-salt buffer washes.The expected recovery of condensin on the bead-bound DNA should be between 20%-40% of the input.The expected recovery of the second DNA is between 5%-10% of the input.Note that condensin will also undergo enzymatic unloading from DNA in the presence of ATP, especially when incubated at elevated ionic strengths. 1The low ionic strength Loading reaction buffer described in this protocol is designed to favor loading over unloading (Figure 3A).The expected results from restriction enzyme treatment of the second DNA capture products are as follows.In an incubation without restriction enzyme, both dig-DNA and pBlueScript second DNA remain in the bead-bound fraction.StuI treatment should release a proportional amount of condensin and pBlueScript DNA into the supernatant, while the two dig-DNA fragments after StuI treatment should remain bead-bound.ScaI treatment in turn should release the linearized pBlueScript DNA into the supernatant, while the intact dig-DNA and condensin should remain in the bead-bound fraction (Figure 3B).

LIMITATIONS
This protocol uses high-salt resistance of the condensin-DNA interaction as an indicator of its topological nature, together with the requirement of a topologically closed DNA substrate.Such a relatively simple setup has advantages, such as minimal protein perturbation, and easy handling.However, this approach does not address where inside the condensin complex the DNA is entrapped.It also cannot distinguish between topological and pseudo-topological entrapment (where the DNA traverses the condensin ring once or twice, respectively).Additional experiments, such as protein interface crosslinking, 6 are needed to investigate these possibilities.
Another limitation of the described bulk biochemical approach is that it cannot distinguish whether a single condensin complex, or multiple condensins together, hold together two DNA substrates.Such information can be obtained when adapting the above-described reagents and reactions to a single molecule microscopy format.The latter method requires fluorescently labeled condensin complexes for visualization. 1 The described protocol is suitable not only for investigating condensin-DNA interactions, but also those of other SMC family members such as cohesin, the SMC5-SMC6 complex, bacterial or archaeal SMC complexes, as well as more distant relatives like the Rad50 complex.While the required

Figure 1 .
Figure 1.Construction of a budding yeast strain that overexpresses fission yeast condensin (A) Schematic of the three integrative plasmids used to transform budding yeast.Note that fission yeast Cnd3 contains introns, only the cDNA sequences are cloned into the plasmid.(B) Western blot to check protein expression after galactose induction.U, uninduced.I, induced.(C) SDS-PAGE analysis followed by Coomassie Blue staining of the bead-bound protein after small-scale IgG pull down of condensin from different colonies after the third transformation.a-Pk HC and LC, a-Pk tag antibody heavy and light chain, respectively.The asterisk highlights colony 5, which yields a stoichiometric subunit composition of condensin following IgG pull down.

1 .
Wake up condensin overexpression strain from 70 C glycerol stock.a. Streak the condensin-expressing budding yeast strain from the À70 C glycerol stock onto a YPD (Yeast Peptone medium supplemented with 2% glucose) agar plate.b.Incubate the plate at 25 C overnight (16 h).

Figure 2 .
Figure 2. Purification of the fission yeast condensin complex (A) SDS-PAGE analysis followed by Coomassie Blue staining of the protein samples taken from the indicated steps of the IgG affinity pull down.(B) Heparin purification step.Left: chromatogram of the heparin purification step.Blue line, absorbance at 280 nm.Yellow line, conductivity.Green line, fractions collected for SDS-PAGE and nuclease activity analyses.Right: SDS-PAGE analysis of the indicated fractions from the heparin elution (top), and the results from a nuclease activity assay of the corresponding fractions (bottom).nc, nicked circular; l, linear; sc, supercoiled circular.Brown line, fractions collected for concentration and further purification.(C) Superose 6 size exclusion step.Left: chromatogram of the Superose 6 purification step.Blue line, absorbance at 280 nm.Yellow line, conductivity.Pink line, fractions collected for SDS-PAGE analysis.Right: SDS-PAGE analysis of the indicated fractions from the Superose 6 elution.Light pink line, the pooled fractions of purified condensin.

Figure 3 .
Figure 3. Second DNA capture reaction using purified condensin (A) Second DNA capture followed by direct sample analysis.Left: schematic of the workflow for a second DNA capture reaction.The first DNA substrate is assembled by tethering digoxygenin-labeled dsDNA (dig-DNA) onto protein G magnetic beads via an a-digoxygenin antibody (a-dig Ab).The first DNA substrate is then incubated with condensin for topological loading.After washing off the excess condensin complex, the beads are further incubated with the second pBlueScript dsDNA plasmid.Right: DNA agarose gel analysis (top) and protein SDS-PAGE analysis (bottom) of the bead-bound fraction after the second DNA capture reaction are shown.(B) Restriction enzyme cleavage to probe the topological nature of the condensin-DNA interaction.Left: a schematic illustrates the expected outcomes of restriction enzyme cleavage using StuI or ScaI, respectively.Right: DNA agarose gel analysis (top) and protein SDS-PAGE analysis (bottom) of both the supernatant (S) and bead-bound (B) fractions after treatment of a 2 nd DNA capture reaction without restriction enzyme (-RE), or the indicated enzymes.

TABLE REAGENT
or RESOURCE SOURCE IDENTIFIER Antibodies a-Pk antibody (unless stated otherwise, dilute 1:10,000 for western blot) Bio-Rad Cat#MCA1360 a-digoxygenin antibody (not used for western, see main text for dilutions or amount to use) (Continued on next page) STAR Protocols 5, 102995, June 21, 2024 Prepare fresh from the stock solutions of each component on the day of the experiment.Keep at 4 C or on ice.Prepare fresh from the stock solutions of each component on the day before purification.Degas by applying vacuum after passing through a 0.22 mm filter.Keep at 4 C or on ice.Prepare fresh from the stock solutions of each component on the day before purification.Degas by applying vacuum after passing through a 0.22 mm filter.Keep at 4 C or on ice.
Prepare fresh from the stock solutions of each component on the day of the experiment.Keep at 4 C or on ice.SDS-PAGE sample buffer (2x)Omit b-mercaptoethanol for long term storage.Store at room temperature (25 C) indefinitely.To use, mix 2 mL 2x SDS sample buffer with 20 mL b-mercaptoethanol.Use within 2 months.(Continuedon next page) PBS-BSA buffer: dissolve 0.5 g BSA in 100 mL PBS.Filter through a 0.22 mm filter.Store at 4 C for one month.
Prepare fresh from the stock solution of each component on the day and keep on ice.
(Continued on next page) Protocol DNA elution buffer: Add 20 mL Proteinase K to 200 mL DNA elution stock buffer.Prepare fresh on the day and keep at room temperature (25 C). 1x CutSmart buffer: dilute the 10x CutSmart buffer (New England Biolabs) in ddH 2 O. Prepare fresh on the day and keep on ice.Note: New England Biolabs has recently changed their CutSmart buffer to use recombinant BSA, called rCutSmart.Both CutSmart and rCutSmart should work for this protocol.